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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
NASA NTRS2007

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Ott, C. M.↗
DOE OSTI2022

On Single-Cell Enzyme Assays in Marine Microbial Ecology and Biogeochemistry

Extracellular enzyme activity is a well-established parameter for evaluating microbial biogeochemical roles in marine ecosystems. The presence and activity of extracellular enzymes in seawater provide insights into the quality and quantity of organic matter being processed by the present microorganisms. A key challenge in our understanding of these processes is to decode the extracellular enzyme repertoire and activities of natural communities at the single-cell level. Current measurements are carried out on bulk or size-fractionated samples capturing activities of mixed populations. This approach – even with size-fractionation – cannot be used to trace enzymes back to their producers, nor distinguish the active microbial members, leading to a disconnect between measured activities and the producer cells. By targeting extracellular enzymes and resolving their activities at the single-cell level, we can investigate underlying phenotypic heterogeneity among clonal or closely related organisms, characterize enzyme kinetics under varying environmental conditions, and resolve spatio-temporal distribution of individual enzyme producers within natural communities. In this perspective piece, we discuss state-of-the-art technologies in the fields of microfluidic droplets and functional screening of prokaryotic cells for measuring enzyme activity in marine seawater samples, one cell at a time. We further elaborate on how this single-cell approach can be used to address research questions that cannot be answered with current methods, as pertinent to the enzymatic degradation of organic matter by marine microorganisms.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

Abstract Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt‐loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme‐coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum , TrmB‐dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii . The ∆ trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name t rm B s u p pressor, or TbsP (a.k.a. “tablespoon”). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild‐type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose‐dependent transcription of gapII , which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii .

Biochemistry & Molecular Biology↗
DOE OSTI2024

Rapid Design and Engineering of Smart and Secure Microbiological Systems (Final Report)

The design and application of successfully engineered biosystems requires an understanding of how engineered microbes will interact with other organisms – either as one-on-one competitors or in the context of microbial consortia. Engineering microorganisms from first principles for non-laboratory, environmental applications is inherently challenging because: (1) engineered systems tend to quickly revert back to their wild-type behaviors; and (2) these systems typically pay a price in reduced fitness, making them uncompetitive against invasive contaminating species (i.e., metabolic burden). For this project, we used a synthetic biology-based strategy to investigate the organization, control, stabilization, and destabilization of natural and engineered microbes. This approach enabled development of (1) single-strain systems capable of detecting and responding to target organisms in the environment; (2) a pipeline for refining and engineering biological constructs in new, non-model host organisms; and (3) improved systems for rapidly designing, engineering, and assaying new biological modules. This coupled approach to safeguard system design is predictable and portable across bacterial species and is focused on microbes that are part of the beneficial plant microbiome. A long-term goal beyond the proposed research is to enable the rational engineering of microbial communities based on first principles of biological design that mimic the smart performance of microorganisms observed in natural systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of small-molecule protein–protein interaction inhibitors for NKG2D

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ + , and CD8 + T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, in this study, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein–protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure–activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

36 MATERIALS SCIENCE↗
DOE OSTI2025

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays. Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant-microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Yadav, Archana↗
NASA NTRS2002

Lipid Biomarkers for Methanogens in Hypersaline Cyanobacterial Mats for Guerrero Negro, Baja California Sur

Analyses of sediments from the vicinity of active methane seeps have uncovered a particular suite of lipid biomarker patterns that characterize methane consuming archaea and their syntrophic, sulfate reducing partners. These isoprenoid biomarkers, largely identified by their anomalously light carbon isotopic signatures, have been a topic of intense research activity and are recorded in numerous methane-rich environments from Holocene to Cenozoic. This phenomenon has implications for depleted kerogens at 2.7 Ga on early Earth (Hinrichs 2002). In contrast, the lipid biosignatures of methane producing archaea are not readily identified through distinct isotopic labels and have received comparably little attention in analyses of archaea in environmental samples. Indeed, environmental analyses generally detect only free archaeal lipids, not the intact, polar molecules found in the membrane of living organisms. As part of the Ames NAI, the 'Early Microbial Ecosystem Research Group' (EMERG) is working to understand microbial processes in the hypersaline cyanobacterial mats growing in the salt evaporation ponds of the Exportadora de Sal at Guerrero Negro, Baja California Sur, Mexico. The aim of this study was to develop methods by which we could identify the organisms responsible for methane generation in this environment. While the ester-bound fatty acids, hopanoids and wax esters provide a means to identify most of the bacterial components of these mats, the archaea which Ere evidently present through genomic assays and the fact of intense methane production (Hoehler et al. 200l), have not been identified through their corresponding lipid signatures. Archaeal core lipids present a number of analytical challenges. The core lipids of methanogens comprise C20, C40 and sometimes C25 isoprenoid chains, linked through ether bonds to glycerol. As well as archaeal (C20), sn-2- and sn-3-hydroxyarchaeol are associated particularly with methylotrophic methanogens. Recently, we have also identified a dihydroxyarchaeol in a hyperthermophilic methanogen (Summons et al. 2002). Additional structural diversity is encoded into the polar head groups that are attached to the glycerol ether cores. The C20 core lipids are readily analyzed by GC-MS as their volatile trimethylsilyl derivatives while compounds with intact polar head groups can only be detected using LC-MS approaches. Our approach was to utilize the alternative of an ether cleavage reagent (BBr3 vs. HI) and a hydride reducing agent to convert all ether lipids to hydrocarbon in order to provide a vertical profile of quantitative information that might be matched to methane fluxes. We have found that while conventional acid hydrolysis and HI treatment will destroy hydroxyarchaeols, molecular information remains intact through use of BBr3 for ether cleavage. This method revealed the presence of traces of biphytane and various ether alkyls associated with some sulfate reducing bacteria within the mat structure. An interesting, and potentially valuable, byproduct of the method utilizing HI was the identification of abundant homohopanoids after superhydride reduction. Evidently present as sulfur-bound diagenetic products these hopanoids are likely cyanobacterial biomarkers in the early stages of diagenetic preservation.

Jahnke, Linda L.↗
DOE OSTI2026

Effect of glutathione-coated Mn-doped ZnS quantum dots on nutrient delivery in basil ( Ocimum basilicum ) plants

Ensuring efficient nutrient delivery while minimizing environmental impacts remains a significant challenge for modern agriculture. Nanotechnology-based fertilizers offer promising strategies to improve nutrient uptake and bioavailability in plants. This research aims to evaluate the use of Glutathione-coated Manganese-doped Zinc Sulfide quantum dots (GSH-ZnS-Mn QDs) as a potential nano fertilizer for basil (Ocimum basilicum). QDs' physicochemical properties were characterized using UV–Vis spectroscopy, photoluminescence, FTIR, and energy-dispersive X-ray spectroscopy, confirming successful Mn doping and glutathione surface functionalization. Basil plants were exposed to different concentrations of GSH-ZnS-Mn QDs under soil and hydroponic conditions. Plant growth parameters, oxidative stress responses, photosynthetic pigments, and macro- and micronutrient uptake were assessed using biochemical assays and inductively coupled plasma optical emission spectrometry (ICP-OES). Elemental uptake, spatial distribution, and zinc speciation were further investigated using synchrotron-based micro-X-ray fluorescence (μ-XRF) imaging and X-ray absorption near-edge structure (XANES) spectroscopy. Results show that exposure to GSH-ZnS-Mn QDs resulted in a concentration-dependent increase in leaf and stem biomass, accompanied by enhanced Zn accumulation in plant tissues. Catalase activity decreased across all tested concentrations, suggesting a shift toward glutathione-dependent antioxidant pathways rather than oxidative damage. Chlorophyll levels exhibited moderate reductions at higher concentrations. The higher increase in macronutrient (K, Ca, and Mg) uptake was reported in plants exposed to 200 ppm of QDs. μ-XRF imaging indicated a selective accumulation of Zn in roots and stems, with partial translocation to leaves. XANES analyses revealed that Zn from QDs was mainly converted into organic Zn species, such as Zn-phytate, Zn-acetate, and Zn-cysteine, indicating transformation and complexation within the plant. The findings demonstrate that a glutathione coating on GSH-ZnS-Mn QDs improves biocompatibility and nutrient delivery efficiency. These results highlight the relevance of surface functionalization in regulating nanoparticle fate, transformation, and nutrient bioavailability, supporting the potential application of GSH–ZnS–Mn QDs as modern nano fertilizers.

Basil↗
NASA NTRS2002

Long Term Manipulations of Intact Microbial Mat Communities in a Greenhouse Collaboratory: Simulating Earth's Present and Past Field Environments

Photosynthetic microbial mat communities were obtained from marine hypersaline saltern ponds, maintained in a greenhouse facility, and examined for the effects of salinity variations. Because these microbial mats are considered to be useful analogs of equivalent ancient marine communities, they offer insights about evolutionary events during the greater than 3 billion year time interval wherein mats co-evolved with Earth's geosphere and atmosphere. Although photosynthetic mats can be highly dynamic and exhibit extremely high activity, the mats in the present study have been maintained for more than one year with relatively minor changes. The major groups of microorganisms, as assayed using microscopic, genetic, and biomarker methodologies, are essentially the same as those in the original field samples. Field and greenhouse mats were similar with respect to rates of exchange of oxygen and dissolved inorganic carbon across the mat-water interface, both during the day and at night. Field and greenhouse mats exhibited similar rates of efflux of methane and hydrogen. Manipulations of salinity in the water overlying the mats produced changes in the community that strongly resemble those observed in the field. A collaboratory testbed and an array of automated features are being developed to support remote scientific experimentation with the assistance of intelligent software agents. This facility will permit teams of investigators to explore ancient environmental conditions that are rare or absent today but might have influenced the early evolution of these photosynthetic ecosystems.

Bebout, Brad↗
NASA NTRS2017

A PCR Based Microbial Monitoring Alternative Method of Detection and Identification of Microbes Aboard ISS

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS) with additional introduction of new microflora occurring with every exchange of crew or addition of equipment and supplies. These microbes are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). As this can be detrimental to astronaut health and optimal performance of ISS systems, monitoring of systems such as ECLSS to include identification of microbial contaminants could prevent adverse effects on human health and life support systems. Current monitoring on ISS is laborious and utilizes culture based methods followed by sample return to Earth for complete analysis. Future, long-distance spaceflight missions will require real-time monitoring capabilities that enable efficient and rapid assessments of the microbial environment allowing for expedited decisions and more targeted response to cope with anomalies. Polymerase chain reaction (PCR), a molecular microbial monitoring method was chosen and numerous PCR instruments investigated for their potential to perform in microgravity conditions. Using ISS as a test bed for PCR verification in microgravity will enable NASA to assess whether molecular based microbiological sensors may be components of reliable, closed-loop life support and habitation systems in spacecraft, enhancing infrastructure capabilities through increased efficiency, reliability, and time savings by enabling sample analysis on orbit. NASA selected the Water Monitoring Suite as one of the rapid spaceflight hardware demonstration activities utilizing a streamlined process to minimize the time required to fly experimental flight hardware. The RAZOR EX (BioFire Defense, Salt Lake City, UT) system was part of the water monitoring suite and is a commercial off-the-shelf (COTS) real-time PCR instrument designed for field work. The RAZOR EX was originally designed for Department of Defense (DoD) under a small business innovative research (SBIR) grant and is ruggedized, compact and provides a rapid, sample to answer in less than an hour. PCR assays using a fluorescent probe were optimized and spiked with known concentrations of DNA (Pseudomonas aeruginosa) ranging from 0.002 to 20 ng. PCR reagents were lyophilized and configured in customized pouches and tested for flight readiness. Three types of water were used to rehydrate the reagents and demonstrate the fidelity of the PCR reaction in microgravity. Molecular grade deionized water served as a control while filtered and unfiltered ISS potable water served to test for chemical or biological inhibitors. All three types were compared to parallel ground test results. Nine tests were run on ISS (3 of each water type) and the critical threshold cycle (Ct) was compared to parallel ground tests completed at Kennedy Space Center, FL and Johnson Space Center, TX. All concentrations of Pseudomonas aeruginosa DNA were detected. A comparison of the Ct produced in real time PCR indicated similarity between flight and ground samples. There appeared to be no significant difference between flight or ground PCR reactions or between any of the three water types. This testing demonstrated the ability to perform molecular testing during spaceflight operations with similar sensitivity. It will allow for future ground development of molecular protocols and minimize the need for spaceflight testing. Future testing will include development of additional targets including environmental and health related organisms.

Christina Khodadad↗
DOE OSTI2026

A Multiplexed Quantitative Analysis of Germline Single Amino Acid Variants by Targeted Proteomics in Nondepleted Human Plasma

Single amino acid variants (SAAVs) in protein sequences are often a direct result of single-nucleotide polymorphisms (SNPs). Certain germline SAAVs have shown biological relevance in different disease conditions but lack precise quantification in circulation, which could hinder functional investigations and progress in biomarker development. Here, we have developed a multiplexed liquid chromatography-selected reaction monitoring (LC-SRM) assay that monitors 5 wild-type and variant peptide pairs (Complement Factor B: CFB-R32Q/R32W, Clusterin: CLU-N317H, Fetuin B: FETUB-K360R, and Kininogen: KNG1-L212P) in nondepleted human plasma. The assay was optimized for imprecision, linearity, stability, and calibration assessments with CVs of under 20%. The wild-type and variant peptide pairs were characterized in a set of healthy individual plasma samples. These target identifications were also validated by SNP genotyping with more than 99% accuracy. For all protein targets, we observed significantly lower concentrations of WT species in the presence variant peptides. In CFB, the concentration of R32Q was significantly lower than its counterpart R32W variant and WT species. Furthermore, our results distinguished phenotypes of homozygosity and heterozygosity of the SAAV presence through direct concentration level characterization. These findings provide some insights into how SAAVs affect quantitative assessments of target peptides. The assay demonstrates a platform for proteogenomic analyses with potential applications in both research and clinical settings.

genetics↗
DOE OSTI2020

A temporal hierarchy underpins the transcription factor–DNA interactome of the maize UPR

Adverse environmental conditions reduce crop productivity and often increase the load of unfolded or misfolded proteins in the endoplasmic reticulum (ER). This potentially lethal condition, known as ER stress, is buffered by the unfolded protein response (UPR), a set of signaling pathways designed to either recover ER functionality or ignite programmed cell death. Despite the biological significance of the UPR to the life of the organism, the regulatory transcriptional landscape underpinning ER stress management is largely unmapped, especially in crops. To fill this significant knowledge gap, we performed a large–scale systems–level analysis of the protein–DNA interaction (PDI) network in maize (Zea mays). Using 23 promoter fragments of six UPR marker genes in a high–throughput enhanced yeast one–hybrid assay, we identified a highly interconnected network of 262 transcription factors (TFs) associated with significant biological traits and 831 PDIs underlying the UPR. We established a temporal hierarchy of TF binding to gene promoters within the same family as well as across different families of TFs. Cistrome analysis revealed the dynamic activities of a variety of cis–regulatory elements (CREs) in ER stress–responsive gene promoters. By integrating the cistrome results into a TF network analysis, we mapped a subnetwork of TFs associated with a CRE that may contribute to UPR management. Lastly, we validated the role of a predicted network hub gene using the Arabidopsis system. The PDIs, TF networks, and CREs identified in our work are foundational resources for understanding transcription–regulatory mechanisms in the stress responses and crop improvement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

SPOTTED-LEAF7 targets the gene encoding β-galactosidase9, which functions in rice growth and stress responses

Abstract β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the nonreducing ends of β-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical β-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Data for "Efficient Mutagenesis and Genotyping of Maize Inbreds Using Biolistics, Multiplex CRISPR/Cas9 Editing, and Indel-Selective PCR"

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

gene editing↗
DOE OSTI2022

2016 Panama ENSO Non-Structural Carbohydrates (NSC), Feb2016-May2016, PA-SLZ, PA-PNM, PA-BCI

Results from Non-Structural Carbohydrate analysis of leaf and branch samples are provided in 2016ENSO_Panama_NSC.xlsx. The metadata files (Metadata_description_2016_ENSO_Panama.docx, File_Submission_Metadata_v1_2016ENSO_Panama_NSC.xlsx), field log (E-Field_Log_2016ENSO_Panama.xlsx), and protocols (ENSO NSC field protocol.pdf, Tropics NSC Assay protocol.pdf) contain additional information. Contact lee@lanl.gov for additional information. VERSION 2 update. The identification of a species from the PNM site has been corrected as follows: the identification of the tree initially identified as Pseudosamanea guachapele (ALBIED) has been revised to Albizia adinocephala (ALBIAD). The updated data package includes revised data, metadata and protocol documents updated to reflect this change.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS1984

Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydrofluoric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.

Ribitol/analogs & derivatives↗
NASA NTRS1998

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

NASA Discipline Environmental Health↗